PROJECT SUMMARY How the intracellular membrane system of eukaryotic cells is configured and maintained is a fundamental problem in cell biology. Deficiencies in this organization often lead to disease. The overarching goal of my laboratory is to define the molecular mechanisms that regulate membrane dynamics, including vesicle biogenesis, organelle trafficking, and protein sorting in the early secretory and endocytic pathways of metazoan cells. We aim to determine how normal membrane transport contributes to cellular homeostasis and understand the molecular basis for disease states that emerge when trafficking pathways are disrupted. Using a combination of model systems, structural biology, biochemistry, genetics, and high-resolution subcellular imaging, our studies focus on two essential trafficking pathways necessary for protein and lipid export from the endoplasmic reticulum (ER) and protein turnover within lysosomes via a multivesicular endosome (MVE) intermediate. Mutations in several factors that regulate these processes have been implicated in cancer, diabetes, immune dysfunction, and neurodegeneration. Thus, deciphering the fundamental principles underlying the regulation of ER export and MVE biogenesis should facilitate the future identification of therapeutic targets for disease intervention. One major focus of my research has been elucidating how the early secretory pathway is organized. Our findings revealed the existence of a conserved membrane interface, which links subdomains on the endoplasmic reticulum (ER) that produce COPII-coated transport carriers to juxtaposed ER-Golgi intermediate compartments (ERGIC). We identified Trk-fused gene (TFG) as a key constituent of this interface and have shown that its inhibition uncouples ER and ERGIC membranes and leads to the isotropic diffusion of COPII-coated carriers, reducing the efficiency of cargo secretion. My second research focus is aimed at understanding the mechanisms that direct the formation of MVEs, which bud intralumenal vesicles (ILVs) into their interior to sequester membrane-associated cargoes from the cytoplasm. Eventual fusion of MVEs with lysosomes results in the degradation of ILVs and their associated proteins, which plays a key role in tumor suppression by governing the capture and sequestration of signaling receptors. Using C. elegans, we have developed a new, highly simplified, and genetically tractable system to investigate how components of the endosomal sorting complex required for transport (ESCRT) machinery enables the movement of endocytosed cargo to MVEs and ILVs in the context of an intact, developing animal. Our future work will continue to capitalize on evolving methodologies to further establish how COPII-mediated transport and ESCRT-mediated MVE biogenesis are properly regulated. A better understanding of these processes will yield key insights into the homeostatic controls that sustain normal protein trafficking in the secretory and endocytic pathways during cell proliferation and differentiation.